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Thesis (M.Sc.)(Genetics--University of Pretoria, 2005. / Title from opening screen (viewed March 28, 2006). Summaries in English and Afrikaans. Includes bibliographical references.

by Joseph Adeleke Adegoke. / Thesis (M.S.)--Florida State University, 1972. / Bibliography: leaves 57-60.

by Karin Sturm. / Thesis (M.S.)--Florida State University. / Bibliography: leaves 64-71.

A dissertation Submitted in fulfilment of the requirements for the degree of Master of Science to the Faculty of Health Sciences at the University of the Witwatersrand, Johannesburg, South Africa. November 2014 / The genome is comprised of a simple quaternary code that serves as the “software” which governs all the complex processes of life. The successful manipulation of this code holds enormous potential for applications involving genome engineering. However, the tools needed to navigate the complex landscape of the genome and efficiently and precisely introduce engineered modifications are lacking. The clustered regularly interspaced short palindromic repeats (CRISPR) adaptive immune system found in prokaryotes functions to recognise and silence foreign pathogenic DNA by double-stranded break (DSB) DNA digestion. The type II CRISPR system of Streptococcus pyogenes has recently been reconstituted to function in mammalian cells as a highly programmable and efficient gene-targeting nuclease platform. Through the heterologous expression of the CRISPR associated (Cas) 9 endonuclease and a small single-guide RNA (sgRNA) molecule, specific gene sequences can be targeted for a DNA DSB. The repair of this targeted DNA damage can be exploited to mutate gene sequences or reconstitute break sites according to a homologous repair template for precise gene modifications. In this study, the capabilities of the CRISPR/ Cas9 system for genome editing was tested by using it to precisely mutate the KRAS proto-oncogene. A panel of five KRAS targeting sgRNAs were designed around a common G>T mutation in codon 12 and characterised. The ability of the CRISPR/ Cas9 system to stimulate DSBs in this region was assessed using the Surveyor Assay, which is able to detect the non-homologous end joining repair of these breaks. A maximum cleavage activity of 6.01 and 2.74% was detected, upstream and downstream of the mutation site, respectively. Simultaneous cleavage by these two sgRNAs was able to successfully introduce a locus-specific micro-deletion. The homology directed repair of these DSBs according to a 90-mer single-stranded oligodeoxynucleotide repair template was shown with the RFLP assay. Analysis of these results implicated guide sequence composition and DSB repair pathway bias as potential factors which may affect the efficiency of desired gene-editing outcomes. These characterised sgRNAs were then applied to generate selectable G>T KRAS mutants. A “dual-cut” strategy, which was designed to overcome gene conversion limitations was employed, and the outcomes were measured with qPCR. The results show that 0.123% of transfected cells were successful recombinants, demonstrating that the use of a “dual-cut” strategy with the CRISPR/Cas9 system was functional and efficient for the generation of knock-in mutants. The CRISPR/Cas9 system has proven to be efficient and robust in modifying the human KRAS locus in various ways. With its modularity and simplicity, CRISPR/Cas9 is a powerful tool that will allow for the modification and interrogation of gene function.

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In the past decade, technologies such as the DNA microarray and ChIP-on-chip have generated a large amount of high-throughput data for biologists. Although these data has provided us systems-level information about gene regulation, a major challenge in systems biology is to derive methodologies that will infer the underlying dynamics and mechanisms of gene regulation. This thesis research is focused on understanding these mechanisms of transcriptional regulation using systems biology approaches. Transcription regulatory networks play an important role in mediating external stimuli and coordinating responses to changing environments. Different methods that infer regulatory interactions directly from microarray data have been developed in the recent past. However, the implicit assumption in these methods that the transcription factor (TF) mRNA expression can be used as a proxy of its activity at protein level is not always correct, due to post-transcriptional and post-translational modifications of TFs. In this study, a method named iARACNe was developed. It uses the inferred TF activities to estimate the regulatory activity between TFs and their targets. The study demonstrated that the accuracy of the inferred networks using this method was greatly improved. Two additional methods, OmniMiner and coEDGi, which allow a better understanding of the physical interactions between TFs and target genes, were developed in this thesis research. OmniMiner detects and predicts the potential binding sites for the TFs of interest, while coEDGi enables identification of common enhancers upstream of co-regulated genes. Compared to other approaches which only allow isolated analyses, the systems biology approaches developed in this research provide an opportunity for biologists to study transcriptional regulations from both functional genomics and regulatory sequence perspectives simultaneously.

Today, malaria remains the biggest killer of the third world, killing over a million people every year, despite intensive research efforts. Carbamoyl phosphate synthetase II (CPSII) is the first and rate-limiting enzyme in pyrimidine biosynthesis of Plasmodium falciparum, the causative agent of malaria. PfCPSII is a unique target for DNAzyme therapy due to the presence of two unique insertion sequences of 700bp and 1800bp that exist within the mature mRNA transcript. Previous studies have demonstrated that exogenous delivery of nucleic acids such as ribozymes and DNAzymes targeting PfCPSII insertion II effectively inhibited the growth of P. falciparum cultures at sub-micromolar levels. The objective of this study was to investigate the insertion sequences within CPSII from rodent malaria species P. berghei, P. chabaudi and P. yoelii in order to further validate the insertions as DNAzyme targets in vivo. In addition, the insertions were isolated from another human malaria parasite, P. vivax. All Plasmodium CPSII genes investigated encoded two highly hydrophilic insertion sequences of similar size and nature, in the precise position seen in PfCPSII. Although these insertions are poorly conserved, border and internal regions of high homology are present. Thirty-one new DNAzymes were designed to target the P. berghei CPSII insertion II region, seventeen of which demonstrated the ability to cleave the target RNA. Of these, four showed significant cleavage activity, with the DNAzyme MD14 cleaving greater than half the target RNA within five minutes. These DNAzymes were then further characterised for kinetic behaviour. Again, MD14 displayed favourable kinetics of cleavage and was chosen as a suitable candidate in an in vivo rodent malaria trial. Analysis of parasitaemia from the MD14 treated mice indicated the administration of MD14 effected a highly statistically significant reduction of parasitaemia, although this reduction was low (6.3%). More efficient DNAzyme delivery methods were investigated in order to improve DNAzyme efficacy and included the novel use of porphyrin conjugated DNAzymes. The porphyrin-conjugated DNAzymes improved uptake into parasitised red blood cells and significantly reduced parasite growth in vitro at nanomolar levels.

Today, malaria remains the biggest killer of the third world, killing over a million people every year, despite intensive research efforts. Carbamoyl phosphate synthetase II (CPSII) is the first and rate-limiting enzyme in pyrimidine biosynthesis of Plasmodium falciparum, the causative agent of malaria. PfCPSII is a unique target for DNAzyme therapy due to the presence of two unique insertion sequences of 700bp and 1800bp that exist within the mature mRNA transcript. Previous studies have demonstrated that exogenous delivery of nucleic acids such as ribozymes and DNAzymes targeting PfCPSII insertion II effectively inhibited the growth of P. falciparum cultures at sub-micromolar levels. The objective of this study was to investigate the insertion sequences within CPSII from rodent malaria species P. berghei, P. chabaudi and P. yoelii in order to further validate the insertions as DNAzyme targets in vivo. In addition, the insertions were isolated from another human malaria parasite, P. vivax. All Plasmodium CPSII genes investigated encoded two highly hydrophilic insertion sequences of similar size and nature, in the precise position seen in PfCPSII. Although these insertions are poorly conserved, border and internal regions of high homology are present. Thirty-one new DNAzymes were designed to target the P. berghei CPSII insertion II region, seventeen of which demonstrated the ability to cleave the target RNA. Of these, four showed significant cleavage activity, with the DNAzyme MD14 cleaving greater than half the target RNA within five minutes. These DNAzymes were then further characterised for kinetic behaviour. Again, MD14 displayed favourable kinetics of cleavage and was chosen as a suitable candidate in an in vivo rodent malaria trial. Analysis of parasitaemia from the MD14 treated mice indicated the administration of MD14 effected a highly statistically significant reduction of parasitaemia, although this reduction was low (6.3%). More efficient DNAzyme delivery methods were investigated in order to improve DNAzyme efficacy and included the novel use of porphyrin conjugated DNAzymes. The porphyrin-conjugated DNAzymes improved uptake into parasitised red blood cells and significantly reduced parasite growth in vitro at nanomolar levels.